1. Technical Field
The present invention is generally related to the use of intrinsically disordered proteins as conformation-switching biosensors, and more specifically related to the use of an alpha synuclein (αS) variant, PG65, together with conformation-sensitive fluorescence to create a molecular probe for rapid, specific and quantitative detection of αS oligomers, which are the major toxic aggregate forms in Parkinson's disease.
2. Prior Art
Aggregation of a synuclein (αS), which is a 140-amino acid, structurally flexible, intrinsically disordered protein (IDP), is central to the pathogenesis of Parkinson's disease (PD).1 αS aggregation involves a molecular process of monomeric αS self-assembling to form αS oligomeric forms followed by further stacking into large fibrillar αS aggregates2. The distinct αS aggregate assemblies display different, yet similar, conformations and exert a varying extent of biological effects.3 Despite its scientific and biomedical importance, the exact nature of αS aggregation is poorly understood, in part due to the limited capability to detect structurally similar yet different αS assemblies specifically and reliably.4 
The non-β amyloid component domain (E61-V95, NAC) of αS is critical in αS self-assembly.5 Within the NAC domain, hydrophobic residues with high β sheet potentials (referred to as HNAC in FIG. 1A) are connected by residues with high turn potentials (referred to as the linker region in FIG. 1A).6 αS monomers are irregularly structured.1 αS oligomers are soluble aggregates which may possess β sheet structures.7 αS oligomers may display different structures depending on size (e.g., low molecular weight (LMW) vs. high molecular weight (HMW) αS oligomers).7a A structural variation can also exist between HMW αS oligomers formed under different incubation conditions.7b αS oligomers can further aggregate to αS fibrils exhibiting cross β sheet structures8 which are similar, but not necessarily identical to β sheet conformations found in αS oligomers.7b,9 In particular, arrangements of β strands in β sheets differ between oligomers and fibrils of αS7b, as is the case with other amyloid proteins.10 
It is widely accepted that β sheet-structured αS oligomers are the major toxic agents in PD7,11. Neither early diagnostics nor disease-modifying drugs are currently available for PD, and specific detection of αS oligomers is quintessential to develop relevant strategies as well as to better understand a molecular basis of PD. A rapid-responsive rather than overnight-long platform is preferred for detection of αS oligomers due to their structurally transient nature.7b,12 Such detection should also benefit high-throughput assays to identify therapeutic agents specifically inhibiting αS oligomerization rather than αS fibrillization. Note that compounds inhibiting αS fibrillization are not necessarily effective at inhibition of αS oligomerization13 and incomplete prevention of αS fibrillization may result in an accumulation of αS oligomers. Quantitative detection of αS oligomers is essential for correct profiling of oligomeric states. Unfortunately, many, if not all, molecular probes responding to αS oligomers are also responsive to αS fibrils.4 Indeed, rapid-responsive, quantitative, αS oligomer-specific detection methods are currently unavailable.
A known peptide probe for diagnostics and therapeutics is shown in European Patent Publication No. EP 2156181 A2 to Cindy S. Orser et al. A known method for the detection of conformationally altered proteins is shown in European Patent No. EP 1700096 B1 to Eugene Davidson et al. Known dyes for analysis of protein aggregation are shown in European Patent Publication No. EP 2507319 A2 to Anatoliy Balanda et al. Known methods, compositions, and kits for detecting protein aggregates are shown in European Patent No. EP 2019836 B1 to Karl J Guegler.
A known peptide probe for rapid and specific detection is shown in US Patent Publication No. US 20130017615 A1 to Jin Ryoun Kim et al. A known sensor device for determining protein aggregation is shown in European Patent Publication No. EP 1556704 A1 to David Allsop et al. Known methods of preventing, treating and diagnosing disorders of protein aggregation are shown in European Patent No. EP 1608350 B1 to Joanne Mclaurin. Known alpha synuclein aggregation assays are shown in European Patent Publication No. EP 1366368 A2 to Daniel Benjamin et al. A known protein stability assay using a fluorescent reporter of protein folding is shown in US Patent Publication No US 20110129935 A1 to Patrick Schaeffer.
Known compounds for use in the detection of neurodegenerative diseases are shown in US Patent Publication No. US 20120251448 A1 to Franz F. Hefti et al. Known antibodies to alpha synuclein are shown in US Patent Publication No. US 20130072663 A1 to Tamie J. Chilcote et al. A known probe for analyzing protein-protein interaction and method of analyzing protein-protein interactions with the use of the same is shown in European Patent No. EP 1229330 B1 to Takeaki Ozawa et al.
Accordingly, there is a need for a conformation-switching fluorescent protein probe for detection of alpha synuclein oligomers and to the use of an alpha synuclein (αS) variant, PG65, together with conformation-sensitive fluorescence to create a molecular probe for rapid, specific and quantitative detection of αS oligomers. It is to these needs and others that the present invention is directed.